Mechanics of human embryo compaction.

The shaping of human embryos begins with compaction, during which cells come into close contact1,2. Assisted reproductive technology studies indicate that human embryos fail compaction primarily because of defective adhesion3,4. On the basis of our current understanding of animal morphogenesis5,6, other morphogenetic engines, such as cell contractility, could be involved in shaping human embryos. However, the molecular, cellular and physical mechanisms driving human embryo morphogenesis remain uncharacterized. Using micropipette aspiration on human embryos donated to research, we have mapped cell surface tensions during compaction. This shows a fourfold increase of tension at the cell-medium interface whereas cell-cell contacts keep a steady tension. Therefore, increased tension at the cell-medium interface drives human embryo compaction, which is qualitatively similar to compaction in mouse embryos7. Further comparison between human and mouse shows qualitatively similar but quantitively different mechanical strategies, with human embryos being mechanically least efficient. Inhibition of cell contractility and cell-cell adhesion in human embryos shows that, whereas both cellular processes are required for compaction, only contractility controls the surface tensions responsible for compaction. Cell contractility and cell-cell adhesion exhibit distinct mechanical signatures when faulty. Analysing the mechanical signature of naturally failing embryos, we find evidence that non-compacting or partially compacting embryos containing excluded cells have defective contractility. Together, our study shows that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.

Mechanical strengthening of cell-cell adhesion during mouse embryo compaction.

Compaction is the first morphogenetic movement of the eutherian mammals and involves a developmentally regulated adhesion process. Previous studies investigated cellular and mechanical aspects of compaction. During mouse and human compaction, cells spread onto each other as a result of a contractility-mediated increase in surface tension pulling at the edges of their cell-cell contacts. However, how compaction may affect the mechanical stability of cell-cell contacts remains unknown. Here, we used a dual pipette aspiration assay on cell doublets to quantitatively analyze the mechanical stability of compacting mouse embryos. We measured increased mechanical stability of contacts with rupture forces growing from 40 to 70 nN, which was highly correlated with cell-cell contact expansion. Analyzing the dynamic molecular reorganization of cell-cell contacts, we find minimal recruitment of the cell-cell adhesion molecule Cdh1 (also known as E-cadherin) to contacts but we observe its reorganization into a peripheral adhesive ring. However, this reorganization is not associated with increased effective bond density, contrary to previous reports in other adhesive systems. Using genetics, we reduce the levels of Cdh1 or replace it with a chimeric adhesion molecule composed of the extracellular domain of Cdh1 and the intracellular domain of Cdh2 (also known as N-cadherin). We find that reducing the levels of Cdh1 impairs the mechanical stability of cell-cell contacts due to reduced contact growth, which nevertheless show higher effective bond density than wild-type contacts of similar size. On the other hand, chimeric adhesion molecules cannot form large or strong contacts indicating that the intracellular domain of Cdh2 is unable to reorganize contacts and/or is mechanically weaker than the one of Cdh1 in mouse embryos. Together, we find that mouse embryo compaction mechanically strengthens cell-cell adhesion via the expansion of Cdh1 adhesive rings that maintain pre-compaction levels of effective bond density.

Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization.

At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts' layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.

Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

Filopodia-like protrusions of adjacent somatic cells shape the developmental potential of oocytes.

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

Cortical softening elicits zygotic contractility during mouse preimplantation development.

Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the second cleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20- to 60-µm range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. We further identify the programmed down-regulation of the formin Fmnl3 as a required event to soften the cortex and expose PeCoWaCo. Therefore, during cleavage stages, cortical softening, mediated by Fmnl3 down-regulation, awakens zygotic contractility before preimplantation morphogenesis.

A theoretical understanding of mammalian preimplantation development.

The blastocyst has long been a hallmark system of study in developmental biology due to its importance in mammalian development and clinical relevance for assisted reproductive technologies. In recent years, the blastocyst is emerging as a system of study for mathematical modelling. In this review, we compile, to our knowledge, all models describing preimplantation development. Coupled with experiments, these models have provided insight regarding the morphogenesis and cell-fate specification throughout preimplantation development. In the case of cell-fate specification, theoretical models have provided mechanisms explaining how proportion of cell types are established and maintained when confronted to perturbations. For cell-shape based models, they have described quantitatively how mechanical forces sculpt the blastocyst and even predicted how morphogenesis could be manipulated. As theoretical biology develops, we believe the next critical stage in modelling involves an integration of cell fate and mechanics to provide integrative models of development at distinct spatiotemporal scales. We discuss how, building on a balanced base of mechanical and chemical models, the preimplantation embryo will play a key role in integrating these two faces of the same coin.

Morphogenesis of the human preimplantation embryo: bringing mechanics to the clinics.

During preimplantation development, the human embryo forms the blastocyst, the structure enabling uterine implantation. The blastocyst consists of an epithelial envelope, the trophectoderm, encompassing a fluid-filled lumen, the blastocoel, and a cluster of pluripotent stem cells, the inner cell mass. This specific architecture is crucial for the implantation and further development of the human embryo. Furthermore, the morphology of the human embryo is a prime determinant for clinicians to assess the implantation potential of in vitro fertilized human embryos, which constitutes a key aspect of assisted reproduction technology. Therefore, it is crucial to understand how the human embryo builds the blastocyst. As any material, the human embryo changes shape under the action of forces. Here, we review recent advances in our understanding of the mechanical forces shaping the blastocyst. We discuss the cellular processes responsible for generating morphogenetic forces that were studied mostly in the mouse and review the literature on human embryos to see which of them may be conserved. Based on the specific morphological defects commonly observed in clinics during human preimplantation development, we discuss how mechanical forces and their underlying cellular processes may be affected. Together, we propose that bringing tissue mechanics to the clinics will advance our understanding of human preimplantation development, as well as our ability to help infertile couples to have babies.

Multiscale analysis of single and double maternal-zygotic Myh9 and Myh10 mutants during mouse preimplantation development.

During the first days of mammalian development, the embryo forms the blastocyst, the structure responsible for implanting the mammalian embryo. Consisting of an epithelium enveloping the pluripotent inner cell mass and a fluid-filled lumen, the blastocyst results from a series of cleavage divisions, morphogenetic movements, and lineage specification. Recent studies have identified the essential role of actomyosin contractility in driving cytokinesis, morphogenesis, and fate specification, leading to the formation of the blastocyst. However, the preimplantation development of contractility mutants has not been characterized. Here, we generated single and double maternal-zygotic mutants of non-muscle myosin II heavy chains (NMHCs) to characterize them with multiscale imaging. We found that Myh9 (NMHC II-A) is the major NMHC during preimplantation development as its maternal-zygotic loss causes failed cytokinesis, increased duration of the cell cycle, weaker embryo compaction, and reduced differentiation, whereas Myh10 (NMHC II-B) maternal-zygotic loss is much less severe. Double maternal-zygotic mutants for Myh9 and Myh10 show a much stronger phenotype, failing most of the attempts of cytokinesis. We found that morphogenesis and fate specification are affected but nevertheless carry on in a timely fashion, regardless of the impact of the mutations on cell number. Strikingly, even when all cell divisions fail, the resulting single-celled embryo can initiate trophectoderm differentiation and lumen formation by accumulating fluid in increasingly large vacuoles. Therefore, contractility mutants reveal that fluid accumulation is a cell-autonomous process and that the preimplantation program carries on independently of successful cell division.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Multiscale morphogenesis of the mouse blastocyst by actomyosin contractility.

During preimplantation development, the mouse embryo forms the blastocyst, which consists of a squamous epithelium enveloping a fluid-filled lumen and a cluster of pluripotent cells. The shaping of the blastocyst into its specific architecture is a prerequisite to implantation and further development of the embryo. Recent studies identified the central role of the actomyosin cortex in generating the forces driving the successive steps of blastocyst morphogenesis. As seen in other developing animals, actomyosin functions across spatial scales from the subcellular to the tissue levels. In addition, the slow development of the mouse embryo reveals that actomyosin contractility operates at multiple timescales with periodic cortical waves of contraction every ∼80 s and tissue remodeling over hours.

aPKCi triggers basal extrusion of luminal mammary epithelial cells by tuning contractility and vinculin localization at cell junctions.

Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi+ cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi+ cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell-substratum contacts. Overall, this study shows that a balance between cell contractility and cell-cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.

Stay hydrated: basolateral fluids shaping tissues.

During development, embryos perform a mesmerizing choreography, which is crucial for the correct shaping, positioning and function of all organs. The cellular properties powering animal morphogenesis have been the focus of much attention. In contrast, much less consideration has been given to the invisible engine constituted by the intercellular fluid. Cells are immersed in fluid, of which the composition and physical properties have a considerable impact on development. In this review, we revisit recent studies from the perspective of the fluid, focusing on basolateral fluid compartments and taking the early mouse and zebrafish embryos as models. These examples illustrate how the hydration levels of tissues are spatio-temporally controlled and influence embryonic development.

Hydraulic fracturing and active coarsening position the lumen of the mouse blastocyst.

During mouse pre-implantation development, the formation of the blastocoel, a fluid-filled lumen, breaks the radial symmetry of the blastocyst. The factors that control the formation and positioning of this basolateral lumen remain obscure. We found that accumulation of pressurized fluid fractures cell-cell contacts into hundreds of micrometer-size lumens. These microlumens eventually discharge their volumes into a single dominant lumen, which we model as a process akin to Ostwald ripening, underlying the coarsening of foams. Using chimeric mutant embryos, we tuned the hydraulic fracturing of cell-cell contacts and steered the coarsening of microlumens, allowing us to successfully manipulate the final position of the lumen. We conclude that hydraulic fracturing of cell-cell contacts followed by contractility-directed coarsening of microlumens sets the first axis of symmetry of the mouse embryo.

[Mechanics of inner cell mass formation]. / Mécanique de la formation de la masse cellulaire interne chez la souris.

During the very first days of mammalian development, the embryo forms a structure called the blastocyst. The blastocyst consists of two cell types: the trophectoderm (TE), which implants the embryo in the uterus and the inner cell mass (ICM), which gives rise to all cells of the mammalian body. Previous works identified how cells differentiate according to their position within the embryo: TE for surface cells and ICM for internal cells. It is therefore essential to understand how cells acquire their position in the first place. During the formation of the blastocyst, cells distort and relocate as a consequence of forces that are generated by the cells themselves. Recently, several important studies have identified the forces and cellular mechanisms leading to the shaping of the ICM. Here, I describe how these studies led us to understand how contractile forces shape the mammalian embryo to position and differentiate the ICM.

Corrigendum: Pulsatile cell-autonomous contractility drives compaction in the mouse embryo.

This corrects the article DOI: 10.1038/ncb3185.

Mechanics of blastocyst morphogenesis.

During pre-implantation development, the mammalian zygote transforms into the blastocyst, the structure that will implant the embryo in the maternal uterus. Consisting of a squamous epithelium enveloping a fluid-filled cavity and the inner cell mass, the blastocyst is sculpted by a succession of morphogenetic events. These deformations result from the changes in the forces and mechanical properties of the tissue composing the embryo. Here, I review the recent studies, which, for the first time, informed us on the mechanics of blastocyst morphogenesis.